Method for placental exfoliation using oxo-arachidonic acid or the like

ABSTRACT

A composition for placental exfoliation and a method for placental exfoliation. The composition for placental exfoliation contains an arachidonic acid derivative or the like, and the method for placental exfoliation comprises the step of administering an arachidonic acid derivative or the like to a mammal after the fetal delivery.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for placental exfoliation andmethods for parturition induction, for example.

2. Background Art

The livestock industry related to farm animals such as cattle is said tobe profitable provided with the successful reproduction. However, in thecase of Hokkaido Japan, the economic loss caused by the deterioration inthe reproductive performance has been preliminarily calculated as 3million yen or more per dairyman on average. As one of the causativefactors of this deterioration in the reproductive performance, technicalinadequacies and accidents related to the parturition can be enumerated.These technical inadequacies and accidents related to the parturitionlead to the increase in the mortality rate of newborns and the loweringin the success rate of conception. Moreover, overworks involved in theattendance in the midnight parturition are also raising big problems atthe production site of the livestock industry where aging of stockraising farmers is in progress.

Under such situations, the parturition-related techniques required bystock raising farmers include techniques for safely inducing the daytimeparturition so as to avoid the attendance in the midnight parturition,and parturition induction techniques for preventing the placentalretention so as to avoid a difficult delivery caused by an oversizedfetus beyond the due date. These techniques bring up various effectssuch as reduced incidence of accident during the parturition,alleviation of mental and physical strain on the manager, appropriatemanagement of the newborn (supply of colostrum without delay), andnormal recovery of a maternal farm animal after parturition.

Examples of the above parturition-related techniques which areconventionally performed include, in cases of cows, the induction ofdaytime parturition by limiting the feeding time and the parturitioninduction using hormones (glucocorticoid or prostaglandin).

The induction of daytime parturition by limiting the feeding time isspecifically performed by feeding during the night time, removing theleftover in the morning, and giving no food during the day time (Wolfe,Modern veterinary practice, Vol. 64, p. 21-23 (1983); and Aoki et al.,Animal Science Journal, Vol. 77, No. 3, p. 290-299 (2006)). However, theconcerned induction yields about 70% probability of the daytimeparturition. Moreover, it is unknown when the parturition after thetreatment occurs. Furthermore, this technique can not be used for cowssuffering from prolonged gestation when calving would not occur evenafter a long-delayed due date.

As shown in FIG. 1, the parturition is achieved in two stages of thefetal delivery and the placental release. In the abovementionedparturition induction using glucocorticoid or prostaglandin, the fetaldelivery serving as the first stage of the parturition is induced.Glucocorticoid promotes the production of prostaglandin andprostaglandin has luteolytic and oxytocic actions. Because of theseactions, the administration of glucocorticoid or prostaglandin inducesthe fetal delivery (Nakahara et al., Kachiku Hansyokushi (Journal ofVeterinary Reproduction), Vol. 21, No. 4, p. 135-140 (1976); Uemura etal., Kachiku Hansyokushi (Journal of Veterinary Reproduction), Vol. 23,No. 3, p. 85-91 (1977); and Aihara et al., Kachiku Shinryo (Journal ofVeterinary Clinic), Vol. 52, No. 11, p. 673-681 (2005)). In theconcerned parturition induction, calving occurs within about 1.5 to 2days post administration of glucocorticoid or prostaglandin, althoughthe incidence of the placental retention is high. Accordingly, theconcerned parturition induction has problems such as a pathologiccondition in which the placenta is not released even though the fetus isdelivered (retained afterbirth).

In this way, there has been no known safe and innovative technique forcontrolling the parturition yet.

SUMMARY OF THE INVENTION

As described above, there has been no known innovative technique forcontrolling the parturition.

On the other hand, it has been conventionally known that the fetaldelivery serving as the first stage of the parturition is caused by theluteolysis and the uterine contraction triggered by prostaglandin.However, researches are not in progress regarding the release ofunneeded placenta serving as the second stage, and the mechanism whichinduces the placental release has not been known yet.

Therefore, in view of the above situations, the inventor of the presentinvention has conducted studies on the mechanism which induces theplacental release at the second stage of the parturition with anobjective of identifying activators of the concerned mechanism andproviding methods for placental exfoliation using these activators.

As a result of intensive studies to solve the above problems, theinventor of the present invention identified the association of matrixmetalloprotease (hereunder, referred to as “MMP”) as a mechanism whichinduces the placental release, and found out that activators of MMP arearachidonic acid (alternatively, referred to as“5,8,11,14-eicosatetraenoic acid”) derivatives and the like. This hasled to the completion of the present invention.

The present invention relates to a composition for placental exfoliationcontaining a compound selected from the group consisting of anarachidonic acid derivative, a linoleic acid, an α-linolenic acid, aγ-linolenic acid, a stearidonic acid, a dihomo-γ-linolenic acid, anω-3-arachidonic acid, an eicosapentaenoic acid, a docosahexaenoic acid,and a salt thereof. The arachidonic acid derivative is exemplified by a12-oxoarachidonic acid (alternatively, referred to as“12-oxoeicosatetraenoic acid”) and metabolites thereof. Moreover,metabolites of the abovementioned highly unsaturated fatty acids (suchas an eicosapentaenoic acid and a docosahexaenoic acid) may also beemployed as active ingredients of the composition for placentalexfoliation.

Moreover, the present invention relates to a kit for parturitioninduction comprising the abovementioned composition for placentalexfoliation. The kit for parturition induction may comprise acomposition for fetal delivery containing a glucocorticoid orprostaglandin.

Further, the present invention relates to a method for placentalexfoliation comprising the step of administering a compound selectedfrom the group consisting of an arachidonic acid derivative, a linoleicacid, an α-linolenic acid, a γ-linolenic acid, a stearidonic acid, adihomo-γ-linolenic acid, an ω-3-arachidonic acid, an eicosapentaenoicacid, a docosahexaenoic acid, and a salt thereof, to a mammal afterfetal delivery. The mammal is exemplified by a cow. The arachidonic acidderivative is exemplified by a 12-oxoarachidonic acid and metabolitesthereof.

Moreover, the present invention relates to a method for parturitioninduction comprising the steps of: administering a glucocorticoid orprostaglandin to a mammal in the gestation period so as to induce fetaldelivery; and administering a compound selected from the groupconsisting of an arachidonic acid derivative, a linoleic acid, anα-linolenic acid, a γ-linolenic acid, a stearidonic acid, adihomo-γ-linolenic acid, an ω-3-arachidonic acid, an eicosapentaenoicacid, a docosahexaenoic acid, and a salt thereof, to the mammal afterthe fetal delivery, so as to induce placental release. The mammal isexemplified by a cow. The arachidonic acid derivative is exemplified bya 12-oxoarachidonic acid and metabolites thereof.

Further, the present invention relates to a composition for MMPactivation containing a compound selected from the group consisting ofan arachidonic acid derivative, a linoleic acid, an α-linolenic acid, aγ-linolenic acid, a stearidonic acid, a dihomo-γ-linolenic acid, anω-3-arachidonic acid, an eicosapentaenoic acid, a docosahexaenoic acid,and a salt thereof. The arachidonic acid derivative is exemplified by a12-oxoarachidonic acid and metabolites thereof.

Moreover, the present invention relates to a method for MMP activationcomprising the step of administering a compound selected from the groupconsisting of an arachidonic acid derivative, a linoleic acid, anα-linolenic acid, a γ-linolenic acid, a stearidonic acid, adihomo-γ-linolenic acid, an ω-3-arachidonic acid, an eicosapentaenoicacid, a docosahexaenoic acid, and a salt thereof, to a mammal. Themammal is exemplified by a cow. The arachidonic acid derivative isexemplified by a 12-oxoarachidonic acid and metabolites thereof.

According to the present invention, the placenta can be released withoutcausing the placental retention. Moreover, according to the presentinvention, the parturition can be safely controlled.

This description includes part or all of the contents as disclosed inthe description and/or drawings of Japanese Patent Application No.2007-246032, which is a priority document of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the office upon request and paymentof the necessary fee.

FIG. 1 is a schematic diagram showing the progress of parturition.

FIG. 2 is a characteristic chart showing the cell exfoliation activityof arachidonic acid derivatives.

FIG. 3 is a characteristic chart showing the cell exfoliation activityof highly unsaturated fatty acids.

FIG. 4 presents photographs showing the manner of parturition.

FIG. 5 presents a photograph showing the MMP activity in releasedplacentas.

PREFERRED EMBODIMENTS OF THE INVENTION

Hereafter, the present invention will be described in detail.

The composition for placental exfoliation of the present inventioncontains a compound selected from the group consisting of an arachidonicacid derivative, a linoleic acid, an α-linolenic acid, a γ-linolenicacid, a stearidonic acid, a dihomo-γ-linolenic acid, an ω-3-arachidonicacid, an eicosapentaenoic acid, a docosahexaenoic acid, and a saltthereof (hereunder, may be referred to as the “active ingredient of thepresent invention” in some cases). The composition for placentalexfoliation of the present invention allows the placenta to beexfoliated and released to the outside from the body of a mammaladministered with the composition after the fetal delivery. Here, theterm “placental exfoliation” refers to a phenomenon in which thematernal placenta and the fetal placenta are separated after the fetaldelivery to release the unneeded fetal placenta out from the uterus. Thesubject to be administered with the composition for placentalexfoliation of the present invention is exemplified by mammals such ashuman, cow, pig, horse, sheep, goat, monkey, dog, and cat, andparticularly preferably cow.

In the present invention, the arachidonic acid derivative refers to a12-lipoxygenase metabolite of an arachidonic acid. The 12-lipoxygenaseis a redox enzyme which adds a molecular oxygen to an unsaturated fattyacid having a cis,cis-1,4-pentadiene structure so as to introduce ahydroperoxide group to a carbon at the position 12. Examples of thearachidonic acid derivative include a 12-oxoarachidonic acid andmetabolites thereof, a 12(S)-hydroperoxyarachidonic acid (alternatively,referred to as “12(S)-hydroperoxyeicosatetraenoic acid”), a12(R)-hydroperoxyarachidonic acid (alternatively, referred to as“12(R)-hydroperoxyeicosatetraenoic acid”), a 12(S)-hydroxyarachidonicacid (alternatively, referred to as “12(S)-hydroxyeicosatetraenoicacid”), and a 12(R)-hydroxyarachidonic acid (alternatively, referred toas “12(R)-hydroxyeicosatetraenoic acid”). In particular, a12-oxoarachidonic acid and metabolites thereof are preferred.

The arachidonic acid derivative can be produced, for example, by achemical synthesis, or by reacting an arachidonic acid with a12-lipoxygenase (O'Flaharty, Biochim. Biophys. Acta, 1994,1201:505-515). Alternatively, commercially available products may beemployed for the arachidonic acid derivative.

Furthermore, the composition for placental exfoliation of the presentinvention may contain a further derivative of a 12-oxoarachidonic acid,a highly unsaturated fatty acid other than an arachidonic acid (such asan eicosapentaenoic acid, a docosahexaenoic acid, an α-linolenic acid, astearidonic acid, and an ω-3-arachidonic acid) or a metabolite thereof,or a precursor of an arachidonic acid (such as a linoleic acid, aγ-linolenic acid, and a dihomo-γ-linolenic acid) as an activeingredient, instead of, or along with, the arachidonic acid derivative.These compounds have a placental exfoliation effect or an MMP activationeffect similar to those of the arachidonic acid derivative. Thesecompounds can be produced, for example, by a chemical synthesis, or byutilizing the extraction from plant bodies or fish oils, or themicrobial fermentation (Osamu Suzuki et al., Kinosei Shishitsu NoShinten (Recent Development of Functional Lipids) (2006)).Alternatively, commercially available products may be employed for thesecompounds.

Examples of the salts of an arachidonic acid derivative, a linoleicacid, an α-linolenic acid, a γ-linolenic acid, a stearidonic acid, adihomo-γ-linolenic acid, an ω-3-arachidonic acid, an eicosapentaenoicacid, and a docosahexaenoic acid include pharmaceutically acceptablesalts. Such salts are exemplified by alkali metal salts such as sodiumsalts and potassium salts.

The composition for placental exfoliation of the present invention ispreferably sterile and may contain one or more pharmaceuticallyacceptable carriers, excipients or diluents or other components. It maycontain various optional components (such as an excipient, a binder, adisintegrator, a surfactant, a lubricant, a flow promoter, a diluent, aflavoring agent, a colorant, and a fragrant agent) for use of normalmedicaments, veterinary medicaments, or the like, in addition to theactive ingredient of the present invention.

Examples of the excipient include celluloses (such as crystallinecellulose), starches (such as rice starch, potato starch, corn starch,wheat starch, and a starch), dextrin, dextran, sugar alcohols (such asmannitol, erythritol, xylitol, maltose, maltitol, and sorbitol),lactose, and glucose.

Examples of the binder include starches, dextrins, powdered acacia,gelatin, hydroxypropyl starch, methylcellulose, carboxymethylcellulosesodium, hydroxypropylcellulose, crystalline cellulose, ethylcellulose,polyvinylpyrrolidone, and macrogol.

Examples of the disintegrator include starches, hydroxypropyl starch,carboxymethylcellulose sodium, carboxymethylcellulose calcium,carboxymethylcellulose, and low substituted hydroxypropylcellulose.

Examples of the surfactant include sodium lauryl sulfate, soybeanlecithin, sucrose fatty acid esters, and polysorbate 80.

Examples of the lubricant include talc, waxes, hydrogenated vegetableoils, sucrose fatty acid esters, magnesium stearate, calcium stearate,aluminum stearate, and polyethylene glycol.

Examples of the flow promoter include light anhydrous silicic acid,dried aluminum hydroxide gel, synthetic aluminum silicate, and magnesiumsilicate.

Examples of the diluent include distilled water for injection, saline,glucose aqueous solution, olive oil, sesame oil, peanut oil, soybeanoil, corn oil, propylene glycol, polyethylene glycol, and ethyl alcohol.

Moreover, the composition for placental exfoliation of the presentinvention may also contain further additional components.

The dosage form of the composition for placental exfoliation of thepresent invention is appropriately selected as required, examples ofwhich include oral agents such as tablets, capsules, granules, finegranules, and powder, and parenteral agents such as injections andsuppositories. The route of administration of the composition forplacental exfoliation of the present invention is appropriatelydetermined in accordance with the dosage form, examples of which includeoral administrations and parenteral administrations (such as intravenousadministration, intramuscular administration, subcutaneousadministration, intrarectal administration, intranasal administration,and sublingual administration).

The frequency of administration, the dose of administration, and thetiming of administration of the composition for placental exfoliation ofthe present invention are not particularly limited, but may beappropriately determined in accordance with, for example, the type, theage, and the body weight of the mammal, and the degree of the symptom.The frequency of administration is, for example in the administration bymeans of the intravenous injection, once to several times (such astwice, three times, four times, and five times) after the fetaldelivery, and preferably once. The dose of administration of thecomposition for placental exfoliation of the present invention is, forexample in cases of cows, 1 to 5 mg/subject per each time, andpreferably 2.0 to 10.0 μg/kg of body weight per each time, in terms ofthe active ingredient of the present invention contained therein.Moreover, the timing of administration is, for example, within 12 hoursor less after the fetal delivery, and preferably within 2 to 6 hours.

The pharmacological evaluation of the composition for placentalexfoliation of the present invention may be carried out either in vitroor in vivo.

The in vitro pharmacological evaluation is exemplified by a method usingcultured fibroblasts derived from the placenta (MMP-producing cells).First, the composition for placental exfoliation of the presentinvention is added to a culture medium of the concerned culturedfibroblasts. After the addition, the cells are cultured for a fixedperiod of time. Then, in cases where a significantly large number ofthese cells were separated from a solid phase (such as the bottom of theculture plate) as compared to the negative control (without the additionof the composition for placental exfoliation of the present invention,for example), the composition for placental exfoliation of the presentinvention can be judged as having the placental exfoliation effect.

The in vivo pharmacological evaluation is exemplified by a method usingcows which have delivered fetuses by means of the single administrationof a prostaglandin. First, the composition for placental exfoliation ofthe present invention is administered to cows after the fetal delivery.Next, after the administration, in cases where the placenta is releasedsignificantly earlier as compared to the negative control (without theadministration of the composition for placental exfoliation of thepresent invention, for example), the composition for placentalexfoliation of the present invention can be judged as having theplacental exfoliation effect.

The composition for placental exfoliation of the present invention canalso be provided as a safe kit for parturition induction (hereunder,referred to as the “kit for parturition induction of the presentinvention”). In particular, the kit for parturition induction of thepresent invention is achieved by combining the composition for placentalexfoliation of the present invention with a composition for fetaldelivery containing either glucocorticoid or prostaglandin. The use ofsuch a kit for parturition induction of the present invention enablesthe induction of both two stages of the fetal delivery and the placentalrelease in the parturition. In the kit for parturition induction of thepresent invention, the composition for placental exfoliation of thepresent invention and the composition for fetal delivery can be providedin the forms of being contained in separate containers or the like.

A kit may contain a sterile composition containing an amount of aglucocorticoid and/or prostaglandin sufficient to induce fetal deliveryof a mammal and a sterile composition comprising a 12-oxoarachidonicacid or a metabolite thereof, or a salt thereof, sufficient to induceplacental exfoliation, and optionally instructions explaining how toadminister or use these compositions to facilitate fetal delivery andplacental exfoliation. Kits may contain single or multiple dosages ofone or both of these sterile compositions, wherein each dosage may be ina separate container, vial, or syringe. Preferably, each dose containsabout 2.0 to 10.0 μg of 12-oxoarachidonic acid or a metabolite thereof,or a salt thereof per kg of body weight of said mammal. For example,where the average weight of a cow is about 500 kg, such a kit maycontain 1 mg to 5 mg of 12-oxoarachidonic acid or a metabolite thereof,or a salt thereof or for or a human female whose average body weight isabout 70 kg, a dosage of 140 ugr to 700 μgr may be used. These dosagesare representative and one of skill in the art may select a higher orlower dosage based on the body weight of the mammal to be treated aswell as other pharmaceutical or therapeutic considerations.

The glucocorticoid or prostaglandin is commercially available fromveterinary pharmaceutical manufacturers (such as Pfizer Inc.).

The dosage form, the route of administration, the frequency ofadministration, and the dose of administration of the composition forfetal delivery may be in accordance with the above description regardingthe composition for placental exfoliation of the present invention.

The timing of administration of the composition for fetal delivery isexemplified by gestation periods from 10 days before the due date tocases of beyond the due date, and preferably a week before the due date,but is not to be limited thereto. That is to say, the administration ofthe composition for fetal delivery is conducted prior to theadministration of the composition for placental exfoliation of thepresent invention.

Meanwhile, the method for placental exfoliation of the present inventioncomprises the step of administering an active ingredient of the presentinvention to a mammal after the fetal delivery. Moreover, the method forparturition induction of the present invention comprises the step ofadministering a glucocorticoid or prostaglandin to a mammal in thegestation period, and the step of administering an active ingredient ofthe present invention to the mammal after the fetal delivery. Hereunder,the method for placental exfoliation of the present invention and themethod for parturition induction of the present invention arecollectively referred to as the “method of the present invention”.

The mammal in the method of the present invention is exemplified bymammals such as human, cow, pig, horse, sheep, goat, monkey, dog, andcat, and particularly preferably cow.

In the method of the present invention, the route of administration, thefrequency of administration, the dose of administration, and the timingof administration of the active ingredient of the present invention, ora glucocorticoid or prostaglandin may be in accordance with the abovedescriptions regarding the composition for placental exfoliation and thekit for parturition induction of the present invention.

According to the present invention described above, the parturition canbe induced at a desired timing (for example, daytime) without causingplacental retention. Moreover, according to the present invention, evenin cases where the parturition would not occur beyond the due date andthe fetus grows so oversized that the difficult delivery is predicted,the parturition can be promptly induced without causing placentalretention. Furthermore, according to the present invention, the uterusis expected to be recovered at an early stage, which is considered tolead to the improvement of the reproductive performance. Moreover, thepresent invention is not only applicable to the control of the date andtime of parturition, but is also applicable to the treatment for thenaturally-occurring placental retention.

Meanwhile, the composition for MMP activation of the present inventioncontains the abovementioned active ingredient of the present invention.Moreover, the method for MMP activation of the present invention is amethod comprising the step of administering the abovementioned activeingredient of the present invention to a mammal. According to thecomposition for MMP activation of the present invention or the methodfor MMP activation of the present invention, a subject requiring the MMPactivation (for example, a patient suffering from a disease such asresidual placenta) can be treated.

Here, the MMP activation refers to a phenomenon in which a peptide of anMMP precursor enzyme (also, referred to as a proenzyme, which is aninactive state) is partially cleaved to express its activity (such as acollagen degrading activity). The MMP to be activated is exemplified byMMP-2, MMP-9, and the like.

The composition for MMP activation of the present invention can beproduced and used in accordance with the above descriptions regardingthe composition for placental exfoliation of the present invention.Moreover, the method for MMP activation of the present invention can becarried out in accordance with the above descriptions regarding themethod of the present invention. The frequency of administration, thetiming of administration, and the period of administration thereof maybe appropriately determined in accordance with, for example, the type,the age, and the body weight of the mammal, and the degree of thesymptom.

The pharmacological evaluation of the composition for MMP activation ofthe present invention is exemplified by the abovementioned in vitromethod using cultured fibroblasts derived from the placenta(MMP-producing cells), or a method using gelatin zymography.

In the method using gelatin zymography, first, the composition for MMPactivation of the present invention is brought into contact withMMP-producing cells. After the incubation for a fixed time, the cellsare lysed to make a protein solution, which is then subjected to anelectrophoresis using a SDS polyacrylamide electrophoresis gelcontaining a gelatin (MMP substrate). After the migration, thetemperature was kept for a fixed time to progress the enzyme reaction,followed by staining of undegraded gelatin remaining in the gel. Incases where a significantly greater amount of the gelatin was degradedas compared to the negative control (without the contact with thecomposition for MMP activation of the present invention, for example),the composition for MMP activation of the present invention can bejudged as having the MMP activation effect.

EXAMPLES

The present invention is hereafter described in greater detail withreference to the following examples, although the technical scope of thepresent invention is not limited thereto.

Example 1 Placental Exfoliation by the Administration of12-oxoarachidonic Acid (1) Search for MMP Activators

The main cause of failure in the placental release during theparturition is failure in the normal separation (exfoliation) betweenthe maternal placenta and the fetal placenta. There are reports showingthat the placenta of cows suffering from placental retention has low orno detected activity of a certain type of MMP, and that the injection ofa collagenase through the umbilical cord into cows suffering fromplacental retention facilitates the placental exfoliation (MMP is also acollagenase), thus it can be assumed that MMP is associated with theplacental exfoliation (Maj et al., Placenta, 1997, 18:683-687; Eiler etal., JAVMA, 1993, 203:436-443). The concerned MMP widely exists as aprecursor enzyme (proenzyme) in vivo, and the enzyme has to be activatedto exert its functions.

Therefore, cultured fibroblasts derived from the placenta (MMP-producingcells) were used to search for MMP activators.

1-1. Candidate Compounds for MMP Activators

Commercially available products were employed for the candidatecompounds for MMP activators in all cases.

1-2. Search for MMP Activators

Since in vivo MMP-2 is produced from fibroblasts, fibroblasts wereprepared from bovine fetal placenta. A fixed amount of cells were seededon the culture plate, and were treated with cortisol after the monolayerformation. Further, the MMP activator candidate compounds were addedthereto. If an MMP activator candidate compound had an MMP activationability, cells were exfoliated from the culture plate and floatedtherein.

The results are shown in FIG. 2 and FIG. 3. In FIG. 2 and FIG. 3, they-axis shows the percentage of exfoliated cells (%) relative to thenon-treatment (0%) while the x-axis shows the concentration (μM) of theadded compound.

The meanings of the abbreviations in FIG. 2 are as follows.

-   “12(S)-HPETE”: 12(S)-hydroperoxyarachidonic acid-   “12(±)-HPETE”: 12(±)-hydroperoxyarachidonic acid (mixture of    12(S)-hydroperoxyarachidonic acid and 12(R)-hydroperoxyarachidonic    acid)-   “12(S)-HETE”: 12(S)-hydroxyarachidonic acid-   “12(R)-HETE”: 12(R)-hydroxyarachidonic acid-   “12oxoETE”: 12-oxoarachidonic acid-   “Ara”: arachidonic acid

Meanwhile, the meanings of the abbreviations in FIG. 3 are as follows.

-   “C16:0”: palmitic acid-   “C18:0”: stearic acid-   “C16:1”: palmitoleic acid-   “C18:1”: oleic acid-   “C18:2”: linoleic acid-   “C18:3”: α-linolenic acid-   “C18:3(γ)”: γ-linolenic acid-   “C18:4”: stearidonic acid-   “C20:3”: dihomo-γ-linolenic acid-   “C20:4”: arachidonic acid-   “C20:4(ω3)”: ω-3-arachidonic acid-   “C20:5”: eicosapentaenoic acid-   “C22:6”: docosahexaenoic acid

The signal transductions for the parturition that have been known so farare centrally mediated by cyclooxygenase derivatives of an arachidonicacid (prostanoid), and thus the first examination was performed in viewof the possibility of MMP activation using the arachidonic acid. Asshown in FIG. 2 and FIG. 3, the arachidonic acid (Ara) was confirmed tohave a cell exfoliation activity (MMP activation ability). On the otherhand, prostaglandin serving as cyclooxygenase derivatives of arachidonicacid had no cell exfoliation activity.

Three types of lipoxygenase pathways are known as other metabolicsystems of arachidonic acid. Therefore, next, respective lipoxygenaseinhibitors were jointly used to examine the reaction, which showed thata 12-lipoxygenase inhibitor inhibited the arachidonic acid-induced cellexfoliation. In this way, it was shown that the arachidonic acid wasconverted by a 12-lipoxygenase to exert its effect. Thus, cellexfoliation experiments were subsequently performed using12-lipoxygenase derivatives (metabolites) of arachidonic acid.

As a result, as shown in FIG. 2, hydroxyderivatives of arachidonic acid(12(S)-hydroxyarachidonic acid (12(S)-HETE) and 12(R)-hydroxyarachidonicacid (12(R)-HETE)) had no cell exfoliation activities, whilehydroperoxyderivatives of arachidonic acid (12(S)-hydroperoxyarachidonicacid (12(S)-HPETE) and 12(±)-hydroperoxyarachidonic acid (12(±)-HPETE))showed weak cell exfoliation activities. Conversely, 12-oxoarachidonicacid (12oxoETE) serving as a ketone derivative of arachidonic acidshowed a cell exfoliation activity at a lower concentration than that ofthe arachidonic acid itself (FIG. 2). That is to say, it was understoodthat the 12-oxoarachidonic acid had a stronger MMP activation ability.

On the other hand, as shown in FIG. 3, some highly unsaturated fattyacids, which belong to the same category as that of the arachidonicacid, showed cell exfoliation activities (medium activity: γ-linolenicacid, eicosapentaenoic acid, and docosahexaenoic acid, and weakactivity: linoleic acid, α-linolenic acid, stearidonic acid,dihomo-γ-linolenic acid, and ω-3-arachidonic acid), all of which werehowever weaker than the activity of 12-oxoarachidonic acid. However,further metabolites of these highly unsaturated fatty acids and12-oxoarachidonic acid might be found to have stronger cell exfoliationactivities. These substances can also be used for promoting theplacental exfoliation.

(2) Placental Exfoliation by the Administration of 12-oxoarachidonicAcid

In order to induce the fetal delivery, a formulation (10 ml) containinga naturally-occurring prostaglandin (50 mg) was intramuscularly injectedinto the hip of a pregnant Holstein cow (dairy cow) (body weight 779 kg)a week prior to the due date. Moreover, a cow after the third calvingwas again administered with the prostaglandin after 12 hours from thefirst injection thereof (evening on the first day).

In the morning on the third day from the administration ofprostaglandin, the pregnant cow delivered a fetus. The mother cow andthe newborn calf were both healthy.

Next, after 4 hours from the fetal delivery, a saline solution (15 ml)containing 12-oxoarachidonic acid (1 mg) was intrajugularly injectedinto the mother cow which had delivered a fetus. After about 6 hoursfrom the injection, the whole amount of the placenta was released fromthe mother cow. FIG. 4 presents photographs showing the manner of suchparturition. The postexperimental mother cow got pregnant again, showingthat the reproductive function was not affected. In cases of thenon-administration of 12-oxoarachidonic acid, the placenta was notreleased from the mother cow, and the placental retention occurred.

(3) MMP Activity in Released Placenta

The MMP activity in released placenta was analyzed. The samples were asfollows: a placenta released from a mother cow of normal parturition(without specific treatment) (expressed as “normal parturition” in FIG.5); a placenta from a mother cow which was subjected to the fetaldelivery induction with a prostaglandin but suffered from placentalretention (expressed as “PG-induced parturition” in FIG. 5); a placentareleased from a mother cow which was subjected to the fetal deliveryinduction with a prostaglandin and the subsequent placental releaseinduction with a 12-oxoarachidonic acid (expressed as “PG-induction+oxo”in FIG. 5); and a placenta from a mother cow which hadnaturally-occurring placental retention (expressed as “placentalretention cow” in FIG. 5).

The MMP activity was measured using gelatin zymography. That is to say,a gelatin serving as an MMP substrate was previously contained in anormal SDS polyacrylamide electrophoresis gel. The placenta samples weresubjected to electrophoresis using the gel, and then SDS was removedtherefrom. The temperature of the gel was kept with a buffer, andthereby MMP active portions in the gelatin were degraded in accordancewith the strength of the activity. After the reaction, the gel wassubjected to the protein staining, by which the MMP active portions inthe gel remained unstained and thus were detected as white bands. Theactivity was detected only for the proenzyme. Therefore, in cases ofsuccess in the MMP activation, the band of the proenzyme activity wouldbe observed weak in the gel. Conversely, in cases of failure in the MMPactivation, the proenzyme is accumulated, making the band strong. Theproenzyme is autoactivated within the gel, and thus is detectable as theactivity. The results are shown in FIG. 5.

As shown in FIG. 5, the placenta released from the mother cow subjectedto the fetal delivery induction with a prostaglandin and the subsequentplacental release induction with a 12-oxoarachidonic acid showed an MMPpattern very close to that of the placenta released from the cow ofnormal parturition, which was apparently different from those of theplacentas from the cow having naturally-occurring placental retentionand the cow having prostaglandin-induced placental retention. This factis considered to be the evidence proving that the administration of12-oxoarachidonic acid activated MMP to induce the placentalexfoliation.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

1. A composition for placental exfoliation containing a 12-oxoarachidonic acid or a metabolite thereof, or a salt thereof.
 2. A kit for parturition induction comprising a composition for placental exfoliation containing a 12-oxoarachidonic acid or a metabolite thereof, or a salt thereof.
 3. The kit for parturition induction according to claim 2, further comprising a composition for fetal delivery containing a glucocorticoid or prostaglandin.
 4. A method for placental exfoliation comprising the step of administering a 12-oxoarachidonic acid or a metabolite thereof, or a salt thereof to a mammal after fetal delivery.
 5. The method according to claim 4, wherein the mammal is a cow.
 6. A method for parturition induction comprising the steps of: administering a glucocorticoid or prostaglandin to a mammal in the gestation period so as to induce fetal delivery; and administering a 12-oxoarachidonic acid or a metabolite thereof, or a salt thereof to the mammal after the fetal delivery, so as to induce placental release.
 7. The method according to claim 6, wherein the mammal is a cow.
 8. A composition for matrix metalloprotease activation containing a 12-oxoarachidonic acid or a metabolite thereof, or a salt thereof.
 9. A method for matrix metalloprotease activation comprising the step of administering a 12-oxoarachidonic acid or a metabolite thereof, or a salt thereof to a mammal.
 10. The method according to claim 9, wherein the mammal is a cow.
 11. A sterile composition comprising 12-oxoarachidonic acid or a metabolite thereof, or a salt thereof and a pharmaceutically acceptable carrier.
 12. A kit comprising: a sterile composition containing an amount of a glucocorticoid and/or prostaglandin sufficient to induce fetal delivery of a mammal and a sterile composition comprising a 12-oxoarachidonic acid or a metabolite thereof, or a salt thereof, sufficient to induce placental exfoliation, and optionally instructions explaining how to administer or use these compositions to facilitate fetal delivery and placental exfoliation.
 13. The kit of claim 12 which contains multiple dosages of one or both of said sterile compositions, wherein each dosage may be in a separate container, vial, or syringe.
 14. The kit of claim 12, which contains a single or multiple dosages of the sterile composition containing a 12-oxoarachidonic acid or a metabolite thereof, or a salt thereof, and each dose contains about 2.0 to 10.0 μg of 12-oxoarachidonic acid or a metabolite thereof, or a salt thereof per kg of body weight of said mammal.
 15. The kit of claim 14, wherein said mammal is a cow and each dose contains 1 to 5 mg of 12-oxoarachidonic acid or a metabolite thereof, or a salt thereof.
 16. The kit of claim 14, wherein said mammal is a human and each dose contains 140 to 700 μg of 12-oxoarachidonic acid or a metabolite thereof, or a salt thereof. 